Vaccinia virus (VV) is the live smallpox vaccine responsible for the successful eradication of smallpox worldwide. This unparalleled success is now being threatened by bioterrorists reintroducing smallpox, against which vaccination is no longer routine. The revival of smallpox vaccination has been countermanded by relatively high incidence of severe adverse events associated with the currently used live VV vaccine. Thus, there is an imminent need to develop alternative and safe approaches to control not only the actual smallpox infection, but also potential complications from the smallpox vaccination. Critical for the development of effective strategies for the control of poxviral infections is to better understand the host's innate immune defense mechanism(s) against poxviruses in vivo. Natural killer (NK) cells represent an important component of the innate immune system and play a critical role in the control of poxviruses in vivo. However, how NK cells are activated in response to poxviral infection and the underlying mechanism(s) responsible for NK cell-mediated control of poxviruses remain largely undefined. We have shown that innate immune recognition of VV is mediated by Toll-like receptor 2 (TLR2) and that activation of the TLR2-MyD88 pathway is crucial to the control of VV in vivo. In the preliminary studies, we have further demonstrated that the TLR2-MyD88 pathway is required for the activation of NK cells upon W infection and their trafficking to the site of infection in vivo, and that stimulation of the TLR2 pathway by W directly on NK cells, but not on accessory cells such as dendritic cells (DCs), is necessary for NK cell activation in vitro. Taken together, these observations suggest a central hypothesis that direct stimulation of the TLR2-MyD88 pathway on NK cells by W is required for NK cell activation, recruitment and function in the control of W in vivo. To test this hypothesis, we will pursue the following five aims: 1) To define the role of direct TLR2 signaling in NK cell activation, trafficking, and function in vivo; 2) To determine the mechanisms underlying TLR2-dependent NK cell activation and trafficking upon VV infection; 3) To identify the W ligand(s) that triggers the TLR2 signaling pathway; 4) To investigate the role of TLR2-dependent NK cell activation in adaptive CD8+ T cell response to W infection; and 5) To study the role of the TLR2 pathway in the activation of human NK cells by VV. We expect this work will lead to a better understanding of mechanisms underlying NK cell activation, trafficking and function in response to VV infection in vivo, which in turn will provide important insights into the design; of novel strategies to effectively activate NK cell function for the control of poxviral infections.